Enzymes are proteins that catalyze
most of the chemical reactions that take place in the body. They make it
possible for chemical reactions to occur at neutral pH and body temperature. The
chemical compound upon which the enzyme exerts its catalytic activity is called
a substrate.

Proteolytic enzymes act on their
natural substrates, proteins and peptides by hydrolyzing one or more peptide
bond(s).

This process is usually highly
specific in the sense that only peptide bonds adjacent to certain amino acids
are cleaved.

Chromogenic substrates are peptides
that react with proteolytic enzymes under the formation of color.

They are made synthetically and are
designed to possess a selectivity similar to that of the natural substrate for
the enzyme.

Attached to the peptide part of the
chromogenic substrate is a chemical group which when released after the enzyme
cleavage gives rise to color. The color change can be followed
spectrophotometrically and is proportional to the proteolytic activity.

The chromogenic substrate technology
was developed in the early 1970s, and has since then become a tool of
substantial importance in basic research.

The majority of chromogenic
substrate applications are found in various clinical fields. In particular they
have been used to generate fundamental knowledge of the mechanisms regulating
blood coagulation and fibrinolysis.

Furthermore, products based on
chromogenic substrate technology have brought a new generation of diagnostics
into the clinical laboratory.

Prothrombin, the natural substrate of Factor Xa, is cleaved by Factor Xa at two positions, each proceeded by the same four amino acid sequence. Factor Xa activity can be determined by the chromogenic substrate S-2222 which is composed of the same amino acids coupled to a chromophore.

Absorption spectrum of a
chromogenic pNA-containing substrate (S) and of pNA.

The hydrolysis of a peptide-pNA
bond in the chromogenic substrates results in the release of pNA which
in turn changes color. Thus the change in absorbance (DA/min)
is directly proportional to the enzymatic activity.